Probing the chemical unfolding and phospholipid binding to the major protein of donkey seminal plasma, DSP-1 by fluorescence spectroscopy

Abstract

Fibronectin type-II (FnII) proteins which constitute the major fraction in the seminal plasma of many mammals, bind to choline phospholipids on the spermatozoa and play a crucial role in sperm capacitation, which enables the sperm to fertilize the egg. In the current study, we have investigated the heterogeneity of microenvironment around the tryptophan residues in the major FnII protein of donkey seminal plasma, DSP-1, in the native state and upon ligand binding employing fluorescence methods. Steady-state and time-resolved fluorescence studies on the quenching of the protein intrinsic fluorescence employing neutral and ionic quenchers revealed that the environment of the tryptophan residues in DSP-1 is less heterogeneous as compared to other homologous seminal plasma FnII proteins PDC-109 (bovine) and HSP-1/2 (equine). The quenching was decreased by ligand binding with choline containing lipids, Lyso-PC and DMPC providing stronger shielding of the Trp residues than the soluble ligand, phosphorylcholine, which is most likely due to segment(s) of the protein penetrating into the hydrophobic interior of the lipid membrane/micelles. Studies on the chaotrope-induced unfolding of DSP-1 revealed that disulfide bonds in the FnII domains of the protein prevent its complete unfolding, and that cooperativity of unfolding is also modulated by polydispersity of the protein.